Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study provides the systematic characterization of the AcINH gene family in pineapple and identifies AcINH3 as a key negative regulator of sucrose metabolism and drought responses. Overall, the study sounds well about the findings. However, I have some suggestions to improve the presentation of this manuscript.

- The Introduction is generally well-organized but overly long and somewhat repetitive, particularly in the sections describing sugar functions and CWIN roles. It would benefit from substantial condensation. The authors are encouraged to streamline the background information, reduce redundancy, and focus more directly on the knowledge gap and the rationale for studying AcINHs in pineapple. 
- In Fig 1(c), please clearly indicate the identities of the other plant species included in the analysis, either in the figure or its legend
- In results, please include the physicochemical characteristics (e.g., gene ID, GRAVY, protein length, molecular weight, pI) of the AcINH proteins to improve the completeness of the analysis.
- In discussion, some statements regarding INH–CWIN interactions and their role in sucrose metabolism are repeated across sections (3.1–3.3). The discussion could be streamlined to reduce redundancy and improve readability.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Drought stress inhibits the growth and yield of pineapple, an important crassulacean acid metabolism (CAM) fruit crop. The sucrose catabolism mediated by cell wall invertase (CWIN) plays an important role in controlling plant growth and development, as well as adaptive responses to abiotic stresses. Invertase inhibitors (INHs) serve as specific post-translational regulators that modulate CWIN enzymatic activity. However, the INH family has not been systematically characterized in pineapple, and its functional roles in mediating sucrose metabolism and drought resistance remain unclear. In this study, three AcINHs were identified from the pineapple genome, followed by comprehensive analyses of their gene structures, phylogenetic relationships, homology characteristics and protein structures and functions and produced significant results. In general, this manuscript was well-written and results are original and significant and data were well presented, I have only following minor comments:

 

Minor Comments:

1. In the end of the abstract and the conclusion part, the authors should stress more about its theoretical and application value of this study;
2. The first letter of each key word should be capital;
3. The Figure 5A and Figure 6A, all mutant lines should be itatical;

4. References need to be updated, there is no references cited from 2025-2026

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript describes a genome-wide identification and further analysis of the AcINH gene family in pineapple including  gene structural features, chromosomal localization, and protein motif compositions.  More detail analysis of AcINH3, including CRISPR- knockout, was performed. AcINH3 involvement in sugar metabolism and drought resistance in pineapple was analyzed. The data obtained can become the basis for determining the mechanisms of sugar metabolism regulation in pineapple and points to AcINH3 as a promising molecular target for genetic improvement of drought resistance in pineapple.

Overall, the text requires minor revision, but the experiment itself does not raise any significant criticism.

The Abstract summarizes the research material

Introduction:

The introduction is structured logically: the authors characterize the main functions of CWIN and INH proteins in plants, describe the currently available functional data and provide a rationale for studying INHs in pineapple

 L 50-55 It is recommended to shorten this text and remove the details of which provinces in China pineapples are grown.

L73-76-  It is recommended to add that the invertase enzymes are usually classified into neutral/alkaline invertases (A/N-INVs) and acid invertases (Ac-INVs), based on their pH, and  Ac-INVs can be further divided into cell wall invertase (CWI) and vacuolar invertase (VI)

 L112-113  … transgenic pineapple calluses with a red phenotype - …transgenic pineapple calluses with a red pigmentation in the adventitious shoots and calluses

Results

L146-148  - and what about motif 6 or 8?  In AcINH2 half of the motfs are absent.

L149- In this study…    -it is recommended to start the phrase with a new paragraph

 L157- provide the designations of all  species abbreviations  on Fig. 1C

 L154- furthermore, AcINH3  could be categorized along with INH1 proteins from other species in group I.  – According to Fig1C AcINH3 forms one subcluster with AcINH2 and SbINH2 (Sorghum bicolor??) and one cluster with other INH2, not INH1/

 The choice of invertase inhibitor proteins of 18 species (Fig.1 and 2)  for comparison is not entirely clear. For example, Zea mays has 4 ZmINHs (according to Chen et al., 2019), but only one was taken for comparison.

 L198- … abundance found in leaves, followed by flowers - abundance found in leaves, followed by flowers (Fig 4a-c)

 L 312 – in vivo- in vivo

Discussion

  It is necessary to provide explanations for the fact that gene expression decreased in the edited lines, although the changes as a result of editing did not affect the promoter region.

L348- reference [27]  is incorrect

 L 348- reference [24]  is also incorrect

Material and Methods

 L434 – Did PEG treatment performed once or every day?

 L444-445. Primers used for the expression analysis (including primers for  internal reference gene) should be added to Suppl.

 References – It is necessary to check the correctness of the references and their compliance with the text

Author Response

Please see the attachment.

Author Response File: Author Response.docx